JP3947247B2 - Method for producing active protein composition - Google Patents

Description

[0001]
[Industrial application fields]
The present invention relates to a method for producing a biologically active plasma protein composition. In particular, the present invention relates to a method for producing a desired protein, comprising a heat treatment for the purpose of inactivating a contaminating virus on the desired protein prepared from plasma. More specifically, the protein prepared from plasma is fragmented with an enzyme, the heat-labile portion is removed, and the desired plasma protein fragment obtained is inactivated by contamination or heating after lyophilization. Provide a way to remove that infectivity. Therefore, the present invention is widely used in fields where the protein fragments heated by the above method have biochemical or medical significance, such as therapeutic drugs and replacement therapy drugs.
[0002]
[Background Art and Problems to be Solved by the Invention]
The plasma fraction preparation is prepared from a pool of many human plasmas as a raw material. In human plasma, it is recognized that viruses such as hepatitis virus and human immunodeficiency virus (HIV) that infect through blood are present, and cases of infection through plasma fraction preparations have also been reported. . Therefore, today, many fractional preparations are produced through an inactivation process of contaminating viruses by heating or the like. Inactivation of contaminating viruses is achieved by applying strong physicochemical treatments, but some proteins that constitute the essential components of plasma fractionated preparations are unstable to these treatments. Therefore, in many cases, a method of freeze-drying the prepared protein (lyophilization heating) or a method of specifically inactivating the virus by heating in the presence of protein stabilizers such as saccharides and amino acids in liquid form is performed. Has been.
[0003]
In the first place, a protein basically consists of an amino acid chain, and a higher-order structure and a subunit structure unique to the protein are formed by the interaction between amino acids. Therefore, treatments that irreversibly destroy higher-order structures or subunit structures are inevitable to have an effect of causing protein denaturation on the protein. The inventor of the present application has a viewpoint that a more powerful virus inactivation can be achieved by previously removing a heat-labile portion from an amino acid residue forming a biologically active protein. I stood up and researched to prove this theory.
[0004]
In view of its importance, the inventor of the present application first studied heating of a lysine-binding fragment of plasminogen and subsequent production method. The reason is as follows.
Plasminogen is a plasma protein with a molecular weight of 80,000 and is an enzyme precursor involved in the coagulation and fibrinolysis system of blood. It undergoes limited degradation by plasmin, urokinase, tissue plasminogen activator (tPA), etc., and is converted to the active form of plasmin, which has the function of degrading fibrin clots. Plasminogen can be divided into a part capable of binding to lysine (lysine binding part) and a part where an active center responsible for the above-mentioned activity exists in the molecule. A lysine-binding fragment has an important role in binding to fibrin, and has recently been proved to have an inhibitory effect on angiogenesis (O'Reilly.MS et al. Angiostatin: a novelangiogenesis inhibitor) That mediates the suppression of metastases by alewis lung carcinoma. Cell, 79 , p.315-328, (1994)). The anti-angiogenic action of lysine-binding fragment suggests that administration of the lysine-binding fragment is effective in suppressing cancer metastasis, which is the biggest problem in cancer treatment. If it can be performed, it is expected that a safe cancer metastasis inhibitor without risk of virus infection or the like can be obtained.
[0005]
Plasminogen can be prepared almost completely in a complete form by using a column with lysine as a ligand from plasma or an alcohol fraction thereof. Plasminogen can stably inactivate contaminating viruses, for example, by heating in the presence of ε-aminocaproic acid (Patent Application Publication No. 35756). However, these stabilizers inhibit the binding of affinity chromatography, and an operation such as dialysis is required to remove the stabilizer for affinity purification of a desired substance. Or the decomposition | disassembly process for obtaining the subsequent plasminogen decomposition | disassembly part is required. In addition, regarding the heating in the lyophilized state, an operation itself requiring a special apparatus such as lyophilization is indispensable, and the presence of a protein stabilizer is often required also in this step. As long as the above conditions are not satisfied, more powerful conditions cannot be added. Therefore, there are various problems as described above in order to isolate the lysine-binding fragment of plasminogen by virus inactivation treatment by the conventional method, and in order to overcome this, some dramatic technique is necessary. It was.
[0006]
[Means for Solving Problems, Structure of the Invention]
The present invention provides a method for producing a biologically active protein. The inventors of the present application separated and decomposed the heat-labile part (active center part) of plasminogen with a proteolytic enzyme, specifically collected only the lysine-binding fragment, and heated it to produce lysine. It has been found that the virus can be selectively inactivated without losing the function of the binding fragment. The present invention has been achieved based on the above findings.
[0007]
The method of the present invention is applicable to many proteins having biological activity intended for administration to humans. However, plasminogen is separated from a plasma protein mixture and selectively decomposed to obtain a desired protein. Obtain a minogen lysine-binding fragment and heat inactivate contaminating virus to produce a plasminogen lysine-binding fragment composition, or prepare fibronectin from a mixture of plasma proteins and selectively degrade it to produce fibronectin. It is particularly suitable for obtaining a heparin-binding fragment and producing the desired fibronectin heparin-binding fragment composition after heat inactivation treatment of contaminating virus.
Hereinafter, the present invention will be described by taking plasminogen as an example of a biologically active protein.
[0008]
The method of the present invention has a great feature in that plasminogen is enzymatically decomposed prior to heat treatment to remove a portion unstable to heat, and then heat treatment is performed. FIG. 1 shows an outline of the present invention. As shown in FIG. 1, plasminogen has a heat-stable part and an unstable part. When plasminogen is heated as it is, the protein in the stable part is also entangled in the unstable part and is dragged to cause heat denaturation. This state does not undergo substrate-specific cleavage of the enzyme, making further preparation impossible. However, if the unstable portion is cut and removed in advance before heating, it becomes tolerant to heating.
[0009]
The procedure of the method of the present invention consists of roughly three steps.
(1) Selective degradation of prepared plasminogen:
Plasminogen is selectively degraded to produce a plasminogen lysine-binding fragment that has biological activity and is subject to heat-inactivation of contaminating virus.
(2) Removal of thermally unstable parts of plasminogen:
The thermally unstable portion generated by the selective decomposition is removed from the solution system.
(3) Inactivation of contaminating virus:
[0010]
Plasminogen, which is a matrix substance of a protein having biological activity, which is an example of the subject of the present invention, can be prepared according to several reported methods, and there are no particular restrictions on the preparation. As a preferred example, it can be prepared from fresh frozen plasma using lysine-conjugated chromatography (lysine chromatography) (Chibber, BAK et al., Plasminogen Methods in Enzymology, 34 , p. 424- 432).
[0011]
Next, the prepared plasminogen is limitedly decomposed into a thermally stable lysine-binding fragment and a thermally unstable fragment. This degradation can be applied, for example, by chemical cleavage with bromocyan as reported in Gross et al. (Gross Eet al., J. Biol. Chem., 237 , p. 1856- (1962)) From the viewpoint of reaction selectivity, selective degradation with an enzyme such as elastase is a preferred embodiment (Davidson JF et al., Raven Press, New York, 3 , p.191-209, (1978)). A reaction ratio suitable for a resin in which elastase is immobilized on a carrier, for example, plasminogen is reacted at an enzyme substrate ratio of 1: 100, and after the post-treatment, the reaction solution is passed through lysine chromatography to collect a flow-through fraction. To do. The plasminogen lysine binding site adsorbed on the resin is eluted with an appropriate elution buffer containing 20 mM aminohexanoic acid and fractionated. If necessary, a protein fraction having a desired molecular weight is obtained by molecular sieve (gel filtration) chromatography of the separated solution.
[0012]
The solution containing the obtained plasminogen lysine-binding fragment is subjected to inactivation of potentially infectious virus by a suitable means. For inactivation of infectious virus, heat inactivation treatment generally used can be applied. Low temperature pasteurization in solution or lyophilized conditions is recommended and requires heating at a temperature of at least 50 ° C. for at least 10 hours. In heating, if necessary, the presence of sugar, amino acid, ε-aminocaproic acid or a salt thereof, and the like can suppress the decrease in the activity of the protein having the desired biological activity. Thus, according to the method of the present invention, a composition containing a plasminogen lysine-binding fragment with an effective and safe guarantee in which a decrease in the activity of the protein due to heating is not observed and the infectious contaminating virus is inactivated. Is provided.
[0013]
A heparin-binding fragment of fibronectin is recognized as a new activity by fragmenting a protein in the same manner as the above-mentioned lysine-binding fragment. Homandberg et al. Prepared a heparin-binding fragment consisting of 29K-da in the molecule after limited degradation of fibronectin with Cathepsin and α-thrombin, and showed that the fragment has an angiogenesis inhibitory effect (Homandberg GA et al., Am. J. Pathol., 120 , p.327-332 (1985)). Fibronectin is also one of the heat-labile substances like the lysine-binding fragment described above. The inventor of the present application prepared a fibronectin heparin-binding fragment from fibronectin according to the method of Homandberg et al. Described above, and heated it in liquid form at 60 ° C. for 10 hours.
[0014]
As a result of heating, the 72K-da fragment obtained by control fibronectin and Cathepsin caused protein denaturation, and the protein recovery after filtration was less than 5%, whereas 29K-da showed no clouding of the liquid. The protein recovery rate was 90% or more. Further, even when α-Thrombin was allowed to act on the heat-denatured precipitates of fibronectin and 72K-da fragment, the recovery rate of the 29K-da fragment was less than 10% of the theoretical value. When the 29K-da fragment before and after heating was added to the proliferation system of vascular endothelial cells and its inhibitory effect was evaluated, the heated heparin-binding fragment showed activity equivalent to that of non-heated.
[0015]
Regarding the application of the patented invention of the present application, plasminogen and fibronectin are exemplified and described among plasma proteins, but the scope thereof is not limited thereto. In other words, in recent concepts of receptors and ligands, in addition to the functions of the entire protein, there are those that are notable for their activity as ligands. The ligand part is a part of the protein, and in the same manner as in this method, the protein may be enzymatically fragmented to prepare the part from plasma protein. When the ligand part is formulated, a heat treatment is carried out, and this is also the case when the same heating as the method provided by the present invention is performed. It is also possible to selectively prepare heat-stable fragments present in a small amount in the living body by heat-treating the unstable fragments and removing them.
[0016]
Hereinafter, in order to deepen the understanding of the present invention, description will be made along examples. However, the present invention is not limited to these examples.
[0017]
【Example】
Example 1
(Preparation of plasminogen)
To 10 liters of fresh frozen plasma, 20 mM benzamidine, 1 mM PMSF, 100 U / ml aprotinin (Tradiol (Bayer)) were added and cold-thawing was performed at room temperature. Thereafter, the suspended matter was centrifuged at 8,000 rpm, 4 ° C. for 20 minutes with a high-speed centrifuge (Tomy Seiko RS-20IV) to obtain a supernatant. The supernatant was passed through a Lysine-Shepharose 4B column (φ5.0 × 30 cm, Pharmacia) equilibrated with 50 mM Tris / 0.5 M NaCl (pH 7.5) at a flow rate of 1.0 ml / min. Further, it was washed with 5 volumes of the same buffer. Thereafter, the buffer was eluted with the same buffer containing 10 mM aminohexanoic acid. The eluate was dialyzed overnight at 4 ° C. against 0.1M ammonium carbonate buffer.
[0018]
Example 2
(Preparation of elastase-Sepharose)
50 mg of elastase (Sigma Tyep IV: From Porcine Pancreas) was dissolved in 0.1 M sodium hydrogen carbonate containing 0.5 M NaCl, and dialyzed against the same buffer at 4 ° C. overnight. CNBr-Activated Sepharose 4 Fast Flow (Pharmacia) was used as the gel for immobilizing elastase, and the coupling was performed at a dose of 5 mg Elastase / ml Gel according to the instruction manual.
[0019]
Example 3
(Preparation of plasminogen elastase degradation product (mini-plasminogen, LBS-I, LBS-II))
Plasminogen prepared by the lysine affinity gel of Example 1 was digested with the elastase prepared in Example 2 according to the method of Davidson et al. (Davidson JF et al., Raven Press, New York, 3 , p.191-209). (1978)), and then the elastase degradation product of plasminogen was separated by lysine affinity gel.
That is, aprotinin 100 U / ml (Tradirol, Bayer) was added to purified plasminogen 10 mg / ml and dissolved in 0.1 M ammonium carbonate. To this, elastase-Sepharose was added so that the enzyme substrate ratio was 1: 100, and the mixture was reacted at 25 ° C. with stirring overnight.
After completion of the reaction, the reaction solution was filtered through a glass filter, and the filtrate was passed through lysine-Sepharose (Pharmacia) equilibrated with 0.1 M ammonium carbonate buffer solution and washed with the same buffer solution. The flow-through fraction (miniplasminogen) was collected with a fraction collector (LKB Redirac E). The lysine-Sepharose binding fraction was eluted with the same buffer containing 20 mM aminohexanoic acid. After the flow-through fraction and the bound fraction (Plasminogen Lysine Binding Site I; hereinafter referred to as LBS-I, Plasminogen Lysin Binding Site II; hereinafter referred to as LBS-II, mixed solution) were pooled, ultrafiltration membranes (manufactured by YM-10 Amicon) ), And dialyzed with 0.1M sodium carbonate and phosphate buffer (pH 7.2) overnight in the air. The concentrated bound fraction was passed through Sephadex G-75 (Pharmacia) equilibrated with 0.1 M ammonium carbonate φ5.0 x 40 cm, and LBS-I (previous fraction), LBS-II (rear fraction) ).
[0020]
Example 4
(Heating of plasminogen lysine-binding fragment)
After lysine-binding fragment (LBS-I fraction) was dialyzed with purified water, 3 ml of each was dispensed into vials, boiled at 100 ° C. and sampled every predetermined time.
[0021]
Example 5
(Evaluation of protein stability (maintaining lysine binding ability))
The LBS-I fraction heated by the method of Example 4 was passed through a Lysine-ligand column, and by examining the amount of the fraction not bound to Lysine-ligand and the bound fraction, The inherent lysine binding ability was compared.
The solution was passed through a Lysine-Sepharose 4 column (φ1.5 × 15 cm) equilibrated with a buffer solution of 50 mM Tris / 0.1 M NaCl / 5 mM EDTA (pH 7.5), washed with the same buffer solution, and then 20 mM aminohexane. Gradient elution was performed with the same buffer containing acid. The absorbance at 280 nm at the time of washing and elution was monitored, and the stability was evaluated by the area ratio of elution.
FIG. 2 shows the elution pattern of heated and non-heated lysine-binding fragments from lysine-Sepharose 4B. As shown in the figure, the amount of protein not adsorbed on lysine-Sepharose by heating at 100 ° C. for 3 minutes is less than 5% of the total, and the elution pattern from the column is the same as that of unheated protein. Met.
[0022]
Example 6
(Evaluation of biological activity of protein)
Regarding the biological activity of the heated lysine-binding fragment, the angiogenesis inhibitory action of the lysine-binding fragment was evaluated using the proliferation ability of vascular endothelial cells according to the method of O'Reilly et al.
That is, according to the method of Folkman et al. (Folkman, J., Haudenschild, CC, and Zetter, BRLong-term culture of capillary endothelial cells.Proc. Natl. Acad. Sci. USA 76 , p. 5217-5221, (1979)). Obtained and managed vascular endothelial cells were prepared at 2,500 cells / ml, and the cells were seeded in a 24-well plate (NUNCLONE, NUNCLONE) at 0.5 ml / well for 24 hours at 37 ° C. in a CO ₂ incubator (CO 2). ₂ concentration 10%). The lysine-binding fragment heated in Example 4 was added to a medium containing 0.25 ml of DMEM, 5% BCS, and 1% kanamycin at 10 μg / ml, and further incubated for 20 minutes. Thereafter, the same buffer containing 1 ng / ml FGF was added to bring the total volume to 500 μl, and the cells were further cultured for 72 hours. As a target, a lysine-binding fragment that was not heated was added. After culturing, the wells were detached with trypsin, and the number of cells was counted.
As a result, when the control to which no lysine-binding fragment was added was taken as 100%, the heated fragment showed 56% and the non-heated fragment showed 58% vascular endothelial cell inhibitory effect. The same vascular endothelial cell proliferation inhibitory effect was confirmed.
[0023]
Example 7
(Protein bioactivity, evaluation in animal tests: comparison of in vivo angiogenesis inhibitory effect) In vivo angiogenesis inhibitory effect was evaluated by the dorsal airsac method using the Abe method (Abe T. et al. al., J. Clin. Invest. 92 , p. 54-61 (1993)). That is, two left and right Millipore chambers were inserted subcutaneously into the back of C57BL6 / J mice, and 3 Lewis mouse lung cancer 3LL-SA1 × 10 ⁶ were injected into this chamber. At this time, tumor cells were not injected into one chamber and used as a control. The heated plasminogen lysine-binding fragment and the non-heated fragment prepared by the method of Example 4 were administered intraperitoneally at 1 mg / kg and reared for 5 days. As the new blood vessels, blood vessels directly under the Millipore chamber were input to the image analyzer, and their areas (occupancy) were compared. The blood vessel occupancy of the plasminogen lysine-binding fragment non-administered group was 15.0 ± 4.3% in the control chamber and 30.3 ± 5.8% in the tumor insertion chamber, whereas the heated fragment-administered group Is 16.1 ± 8.4% in the control chamber and 20.6 ± 7.7% in the tumor insertion chamber, and the non-heated fragment administration group is 19.8 ± 10.4% in the tumor insertion chamber. It was 22.3 ± 9.0%, and angiogenesis derived from the tumor was suppressed.
[0024]
Example 8
(Protein biological activity, evaluation in animal tests: comparison of growth inhibitory effects of lung metastases)
After transplantation of 10 ⁶ cells of Lewis lung cancer (LL / 2) subcutaneously in the back of C57BL6 / J mice, the tumor was excised when the weight reached 500-700 mg, and further bred for 14-17 days. Thereafter, the heated plasminogen lysine-binding fragment and the non-heated fragment prepared by the method of Example 4 were daily administered into the abdominal cavity of the mouse at 1 mg / kg, and further reared for 7 to 10 days. Metastatic growth was compared by measuring the number and weight of lung metastases. As a control, 100 μl of physiological saline was administered instead of the plasminogen lysine-binding fragment. The lung weight was 0.24 ± 0.06 g in the non-heated fragment-administered group, whereas the heated fragment was 0.25 ± 0.12 g, compared to 0.59 ± 0.43 g in the control group. Tumor growth was suppressed.
[0025]
Example 9
(Virus inactivation test)
1/10 amount of Pseudorabies Virus 10 ^8.5 TCID50 / ml was added to the lysine-binding fragment solution prepared in Example 4 and boiled on a water bath for 3 minutes. The infectious titer was measured using Vero cells and the 50% infection end point (TCID50) method.
Before heating, a solution containing 10 ^7.5 TCID50 / ml virus was 10 ^0.5> TCID50 / ml after heating at 100 ° C. for 3 minutes, confirming that the virus was rapidly inactivated.
[0026]
Example 10
(Evaluation in fibronectin)
1. Fibronectin to prepare <br/> material of fibronectin fragments reported such Homandberg (Homandberg GA et al., Arch. Biochem. Biophys. 238, p.652-663 (1985)) was prepared as described in. Human plasma was purified with gelatin Sepharose and partially reduced. After reacting 0.8 μg / ml Catepsin D (manufactured by Sigma) and 1 mg / ml fibronectin for 3 hours at 30 ° C. with 0.1 M borate buffer (pH 3.7), the reaction was terminated, and gelatin Sepharose was added. The binding fragment (72K-da molecule) was recovered by passing through the solution. The bound fragment was dialyzed with 50 mM NaCl / 20 mM Tris buffer (pH 7.4) and reacted with 1 U α-Thrombin, and further decomposed into 29 K-da fragment and 50 K-da fragment. The 29K-da fragment was obtained as the unbound fraction of gelatin Sepharose.
[0027]
2. Fibronectin and heating <br/> fibronectin and fibronectin fragments of fibronectin fragment (72K-da, 29K-da ) after dialysis in saline, 10 hours at 60 ° C., and heated in liquid form. The heating condition at 60 ° C. for 10 hours is a condition that can prevent the transmission of viruses such as hepatitis virus with human serum albumin. Each heated specimen was centrifuged at 3,000 rpm, filtered through a membrane filter (MILEX-HA 0.45 μm: manufactured by Millipore), and the recovery rate was determined by measuring the amount of protein.
The 72K-da protein fragment after heating prepared by the above method was further decomposed with α-Thrombin according to the method of Furie et al. To prepare a 29K-da protein fragment (Furie MB et al., J. Biol. Chem. , 255 , p.4391-4394 (1980)).
[0028]
3. Heating results denatured <br/> heating of proteins by, fibronectin and 72K-da fragments cause protein denaturation, protein recovery after filtration while was less than 5%, 29K-da is the liquid No white turbidity was observed, and the protein recovery rate was 90% or more. Further, even when α-Thrombin was allowed to act on the heat-denatured precipitates of fibronectin and 72K-da fragment, the recovery rate of the 29K-da fragment was less than 10% of the theoretical value.
[0029]
4. Fibronectin fragment according to (29K -da) method described in Evaluation <br/> Example 6 before and after heating of the activity of the determination vascular endothelial cell growth inhibitory effect of heating the front and rear of the protein fragment (29K-da) did. 29K-da before and after heating was added to the proliferation system of vascular endothelial cells shown in Example 6 and the inhibitory effect was evaluated. As a result, the heated heparin-binding fragment showed activity equivalent to that of non-heated.
[Brief description of the drawings]
FIG. 1 is a diagram showing the concept of the present invention.
FIG. 2 is a diagram showing the ability of a plasminogen lysine-binding fragment to bind to lysine before and after heating.

Claims (2)

(a) separating and removing a thermally unstable portion in the protein molecule from a plasminogen- or fibronectin-containing solution by a method based on physical, chemical or enzymatic proteolysis; and (b) said step ( The product after removal of the heat labile part of a), ie, a lysine-binding fragment in plasminogen and a solution containing a heparin-binding fragment in fibronectin under conditions sufficient to inactivate all infectious contaminating viruses. A method for producing a protein composition containing a plasminogen lysine-binding fragment or fibronectin heparin-binding fragment substantially free of infectious contaminating virus, comprising a virus inactivation step of heat treatment. The plasminogen lysine-binding fragment or fibronectin heparin-binding fragment-containing protein according to claim 1, wherein a sugar, an amino acid, or ε-aminocaproic acid or a salt thereof is selectively allowed to coexist during the heat treatment step. A method for producing a composition.
more than

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